negative regulation of tnf by zfp36 1 negative feed-forward

Negative regulation of TNF by ZFP36

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Negative feed-forward control of TNF by tristetraprolin (ZFP36) is limited by the mitogen-activated protein kinase phosphatase, DUSP1: Implications for regulation by glucocorticoids

Suharsh Shah, Mahmoud M. Mostafa, Andrew McWhae, Suzanne L. Traves, Robert Newton1

From the Airways Inflammation Research Group, Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada, T2N 4Z6.

*Running title: Negative regulation of TNF by ZFP36

1To whom correspondence should be addressed: Dr. Robert Newton, Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta, T2N 4N1, Canada. Tel: 001 403 210 3938. Fax: 001 403 270 8928. Email: [email protected]

Key Words: Glucocorticoid, Inflammation, MAPK, DUSP1, ZFP36, TNF, Feed-forward control, Anti-inflammatory Background: ZFP36 negatively regulates AU-rich element-containing mRNAs. Results: DUSP1 silencing increases ZFP36 expression and enhances negative feed-forward control of TNF. Conclusion: MAPK inhibition initially attenuates TNF mRNA, but reduced feed-forward control subsequently produces an increase. Significance: Understanding such networks is essential to develop novel anti-inflammatory therapies or understand TNF repression by glucocorticoids, which may not require DUSP1 or ZFP36 expression.

ABSTRACT Tumor necrosis factor α (TNF) is central to inflammation and may play a role in the pathogenesis of asthma. The 3’–untranslated region of the TNF transcript contains AU-rich elements (AREs) that are targeted by the RNA-binding protein, tristetraprolin (ZFP36), which is itself up-regulated by inflammatory stimuli, to promote mRNA degradation. Using primary human bronchial epithelial (HBE) and pulmonary epithelial A549 cells, we confirm that interleukin-1β (IL1B) induces expression of dual-specificity phosphatase 1 (DUSP1), ZFP36 and TNF. While IL1B-induced DUSP1 is involved in feedback control of MAPK pathways, ZFP36

exerts negative (incoherent) feed-forward control of TNF mRNA and protein expression. DUSP1 silencing increased IL1B-induced ZFP36 expression at 2h and profoundly repressed TNF mRNA at 6h. This was partly due to increased TNF mRNA degradation, an effect that was reduced by ZFP36 silencing. This confirms a regulatory network, whereby DUSP1-dependent negative feedback control reduces feed-forward control by ZFP36. Conversely, while DUSP1 over-expression and inhibition of MAPKs prevented IL1B-induced expression of ZFP36, this was associated with increased TNF mRNA expression at 6h, an effect that was predominantly due to elevated transcription. This points to MAPK-dependent feed-forward control of TNF involving ZFP36-dependent and –independent mechanisms. In terms of repression by dexamethasone, neither silencing of DUSP1, ZFP36, nor both together, prevented the repression of IL1B-induced TNF expression thereby demonstrating the need for further repressive mechanisms by anti-inflammatory glucocorticoids. In summary, these data illustrate why understanding the competing effects of feedback and feed-forward control is relevant to the development of novel anti-inflammatory therapies.

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.697599The latest version is at JBC Papers in Press. Published on November 6, 2015 as Manuscript M115.697599

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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The pro-inflammatory cytokine, tumor necrosis factor (TNF) is induced by various extracellular stimuli and plays essential roles in host responses to infection and injury. However, increased TNF expression is also associated with the pathogenesis of chronic inflammatory disorders, including rheumatoid arthritis, inflammatory bowel disease and asthma (1). Indeed, TNF expression is tightly regulated at a molecular level by transcriptional and post-transcriptional mechanisms (2). While transcriptional control involves recruitment of factors, such as nuclear factor (NF)-κB, to the TNF promoter, post-transcriptional regulation is conferred via multiple copies of the adenylate-uridylate-rich element (ARE) (3), AUUUA, located in the 3’-untranslated region (UTR) of the TNF mRNA (4). Such regions are critical for regulating message stability and are targeted by several RNA binding proteins, including tristetraprolin (TTP; zinc finger protein (ZFP) 36), human antigen R (HuR; ELAVL1), adenine-uridine-rich element RNA-binding factor-1 (AUF1; HNRNPD) and K-homology domain splicing regulatory protein (KHSRP) (5-9). These factors may compete for ARE binding and can variously promote or reduce mRNA stability (4;10). For example, ZFP36 negatively controls TNF expression by promoting mRNA deadenylation and degradation with consequent reductions in TNF biosynthesis (11-13). Similarly, ZFP36 is an established negative regulator of other ARE-containing mRNAs, including cyclooxygenase-2 (PTGS2), colony stimulating factor (CSF) 2, interleukin (IL) 6 and IL8, and mice lacking ZFP36 develop severe and chronic inflammation (8;10).

ZFP36 expression is rapidly induced by multiple pro-inflammatory stimuli, including IL1B or lipopolysaccharide (LPS) in various cells, including macrophage, fibroblasts and A549 pulmonary epithelial cells (14-17). Given the ability to reduce the expression of ARE-containing mRNAs, this means that ZFP36 is a negative (incoherent) feed-forward regulator of inflammatory gene expression (Fig. 1). While increasing ZFP36 reduces the expression of inflammatory genes, ZFP36 protein expression is itself highly dependent on mitogen-activated protein kinase (MAPK) activation (16;18). Following pro-inflammatory stimulation, ZFP36 protein appears initially as a ~40 kDa protein, which becomes phosphorylated and migrates at ~45-kDa on SDS-PAGE (16;19). Phosphorylation is suggested to enhance ZFP36 stability and to promote targeting of ARE containing transcripts (19). However, such a MAPK-dependent negative feed-

forward regulatory loop suggests that MAPK activation may act to reduce the expression of ARE-containing genes via increased ZFP36 activity (Fig. 1). Conversely, reducing MAPK activity may produce opposing effects and could, by reducing negative feed-forward control, promote expression of ARE-containing mRNAs. This scheme is further complicated by the fact that MAPKs are subject to feedback inhibition via a number of processes, including up-regulation of the dual-specificity MAPK phosphatase, DUSP1, which is itself dependent on MAPK activation (20-23). Thus pro-inflammatory stimuli, including IL1B and LPS, increase DUSP1 expression to dephosphorylate and inactivate MAPKs (Fig. 1). In A549 cells, knockdown of IL1B-induced DUSP1 expression transiently increased the appearance of phosphorylated MAPKs and this increased the expression of inflammatory mRNAs at 1 h post-IL1B (24). However, 6 h post-IL1B, this loss of DUSP1 decreased the expression, relative to control, of multiple inflammatory mRNAs. This observation is consistent with the concept that MAPKs may increase ZFP36 expression to subsequently down-regulate ARE-containing mRNAs and is tested in the current study (Fig. 1).

In the context of glucocorticoids, reduced expression of multiple inflammatory genes is central to anti-inflammatory activity (25). However, increased DUSP1 expression is often considered as a key anti-inflammatory effector mechanism (23;26), yet this appears inconsistent with the above idea that MAPKs increase ZFP36 to increase feed-forward control of inflammatory genes. Certainly, glucocorticoids do induce DUSP1 expression, including in A549 cells (27;28). This does reduce MAPK activity and this does play a role in the repression of at least some inflammatory genes in vitro and in vivo (24;29). However, in addition to DUSP1, glucocorticoids induce expression of multiple effector genes and this may lead to redundant actions (30). Indeed, ZFP36 is modestly up-regulated by glucocorticoids in the human airway epithelial cells as well as in pulmonary A549 and bronchial BEAS-2B epithelial cells and in the airways following glucocorticoid inhalation (Leigh R. et al. unpublished data) (16;31;32). Furthermore, a role for ZFP36 in the repression of inflammatory gene expression is indicated (31;33). Given interest in therapeutically targeting MAPK pathways in inflammatory disease, and the fact that glucocorticoids induce DUSP1 to reduce MAPK activity, we have used TNF as a model ZFP36 target gene to explore the relationship(s) between the


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regulation of MAPK activation by DUSP1 with the expression of ZFP36 and effects on downstream gene expression. EXPERIMENTAL PROCEDURES

Gene nomenclature – Official Human Genome Organization (HUGO) gene nomenclature committee gene symbols have been used for all genes and gene products.

Cell culture and drugs – A549 cells were grown in complete DMEM medium (Gibco Life technologies) containing 10% fetal calf serum and 2.0 mM L-glutamine at 37°C in 5% CO2/95% air. HBE cells, isolated from non-transplanted normal human lungs obtained using the tissue retrieval service at the International Institute for the Advancement of Medicine, Edison, NJ, USA, were cultured in bronchial epithelial cell growth medium (BEGM; Lonza, Allendale, NJ) as previously described (34). Prior to experiments, cell growth was arrested by incubating the cells in serum-free medium overnight. At this point cell counts from four independent experiments indicates that there were 1.90 ± 0.05 x 106 cells/well (6 well plates of A549 cells), or 6.1 ± 0.53 x 105/well (6 well plates of HBE cells) and the medium was switched to fresh serum-free medium containing IL1B (1 ng/ml) and/or experimental drugs. IL1B (R&D systems) was dissolved in phosphate-buffered saline (PBS) plus 0.1% bovine serum albumin (BSA) (both Sigma). Dexamethasone (Sigma) was dissolved in Hanks’ balanced salt solution (Sigma) and SB203580 (Calbiochem), (JUN N-terminal kinase) JNK inhibitor 8 (Calbiochem) and UO126 (Calbiochem) were dissolved in DMSO to final concentrations of <0.1%.

Western blot analysis - Western blotting was carried out as described (35). Size-fractionated proteins were transferred to nitrocellulose membranes and probed with rabbit antibodies to DUSP1 (M-18, sc-1102), ZFP36 (H-120, sc-14030) (both from Santa Cruz Biottechnology), uncleaved ~25 kDa TNF (ab66579) (Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4699-9555(ST)) (AbDSerotec) followed by washing and incubation with horseradish peroxidase-linked secondary immunoglobulin (Dako/Jackson ImmunoResearch Laboratories). Immune complexes were detected by enhanced chemiluminescence (GE Healthcare BioSciences) and exposure to X-ray film.

Enzyme-linked Immunosorbent Assay (ELISA) - ELISA for TNF was performed on 100 μl of supernatant using DuoSet ELISA kits (R&D

Systems). For HBE cells, TNF release was determined following concentration of 900 μl of cell supernatant to less than 100 μl using Corning Spin-X® UF 500 Concentrator 5000 (Cat# 431477) columns. Concentrated supernatants were adjusted to final volume of 110 µl using 1% w/v BSA/PBS and ELISA for TNF performed using 100 µl of concentrated supernatant. Cell-associated TNF in A549 cells was detected by lysing the cells in 100 µl of 1x firefly luciferase assay buffer (Biotium) containing 1 × CompleteTM protease inhibitor mixture and phosphatase inhibitors (50 mM NaF, 2 mM Na3VO4 and 20 mM Na4O7P2) followed by 1 freeze-thaw cycle. ELISA for TNF was carried out using 50 µl of cell lysates diluted with 50µl of 1% w/v BSA/PBS. Standard curves were generated using the same firefly luciferase assay buffer BSA/PBS mix.

RNA Isolation, cDNA Synthesis, and SYBR Green Real Time PCR - Total RNA was extracted using the Rneasy mini kit (Qiagen) and 0.5 µg was used to produce cDNA as described (36). Resultant cDNA was diluted 1:4 with RNase-free water and PCR was carried out on 2.5 µl of cDNA using SYBR GreenER mastermix (Invitrogen) with a StepOnePlus™ PCR system (Applied Biosynthesis). Relative cDNA concentrations were derived from standard curves generated by serial dilution of an IL1B-treated sample. Amplification conditions were: 50oC, 2 min, 95oC, 10 min, then 40 cycles of 95oC, 15 s, 60oC, 1 min. Primer specificity was assessed by dissociation (melt) curve analysis: 95oC, 15 s, 60oC, 20 s followed by ramping to 95oC over 20 min.

Adenoviral Infection – As described previously (35), A549 cells at ~70% confluence were incubated with the indicated multiplicity of infection (MOI) of DUSP1-expressing adenoviral vector (Ad5-DUSP1) (Seven Hills Bioreagents) or a GFP (green fluorescent protein)-expressing vector (Ad5-GFP) (Qbiogene) for 24 h in a serum containing medium. Before further treatments, cells were incubated over-night in serum-free medium.

Analysis of Unspliced Nuclear TNF RNA – Unspliced nuclear RNA, or nascent transcript, accumulates transiently in the nucleus following transcriptional activation and may be measured as a surrogate of transcription rate (28;35). Unspliced nuclear RNA was analyzed using SYBR-green primers that crossed the exon 3/intron 4 junction for TNF. Expression was normalised to the abundant small nuclear RNA, U6. Since these primer sets detect both unspliced RNA and genomic DNA, the signal due to the contaminating genomic DNA was assessed in each sample. Each RNA sample was


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subject to reverse transcription, both in the presence and the absence of reverse transcriptase. The presence of an amplification product in the reverse transcription-negative samples was attributed to genomic DNA contamination and samples with greater than 10% genomic contamination for U6 were excluded from further analysis. RNA extraction, cDNA synthesis, and SYBR Green real-time PCR were carried out as described above. Primer sequences were: Unspliced nuclear TNF (forward, TCT CGA ACC CCG AGT GAC A; reverse, CAT CAG CCG GGC TTC AAT) and U6 (forward, AAT TGG AAC GAT ACA GAG AAG ATT AGC; reverse, GGA ACG CTT CAC GAA TTT GC).

siRNA-mediated Gene Silencing – A549 cells were grown in 12 well plates to ∼60–70% confluence and transiently transfected with 1 ml serum-free medium containing DUSP1 or ZFP36 or Lamin A/C siRNA (LMNA) (control siRNA) at a final concentration of 25 nM. Each siRNA was mixed with lipofectamineTM RNAiMAX (1 μl of 1 μg/μl) (Invitrogen) in 100 μl of serum-free DMEM and incubated at room temperature for 30 min prior to dilution to 1 ml and addition to cells. After 24 h the medium was changed to fresh serum-free medium prior to cytokine and drug treatments. Sequences for siRNA targeting were: DUSP1 siRNA 1 (SI00374801; 5′-TAG CGT CAA GAC ATT TGC TGA-3′); DUSP1 siRNA 2 (SI00374808; 5′-CTG TAC TAT CCT GTA AAT ATA-3′) (all Qiagen); LMNA siRNA (control siRNA) (5′-AAC TGG ACT TCC AGA AGA ACA -3′) (Qiagen); ZFP36 siRNA 1 (5′ -ACC GAC GAT ATA ATT ATT ATA-3′), ZFP36 siRNA 2 (5′-ACG ACT TTA TTT ATT CTA ATA-3′) (all Qiagen). Since the expression of ZFP36 and TNF induced by IL1B or IL1B plus dexamethasone was unaltered by LMNA siRNA (data not shown), treatment with IL1B or IL1B plus dexamethasone in the absence of LMNA siRNA was excluded from further analyses.

Data Presentation and Statistical Analysis – GraphPad Prism 5 software was used for all statistical analyses. All data are plotted as means ± SE. One-way ANOVA with a Bonferroni post test was used for comparing 5 or fewer comparisons. Since the Bonferroni post test gives high and increasingly inappropriate false negative rates (i.e. type II or β error) for greater than five comparisons, ANOVA with Newman-Keul multiple comparison test was used for greater than five comparisons, as is recommended for greater power in hypothesis testing (Prism 5, Graphpad Software). ANOVA with a Dunnett's post test was used for comparisons

against a single control column. Two-tailed, paired Student t test was used for comparing two treatment groups. RESULTS

Characterization of TNF expression in the presence of IL1B and dexamethasone in A549 cells - As a prelude to analyzing the regulation of TNF expression by DUSP1 and ZFP36, we characterised the expression of TNF following IL1B treatment in the absence and presence of the synthetic glucocorticoid, dexamethasone. IL1B rapidly increased TNF mRNA (Fig. 2A), which reached a peak at around 2 h post stimulation and then declined steeply towards basal levels over the following 4 h. This was accompanied by a transient elevation of unspliced nuclear TNF RNA and suggests that rapid enhancement of TNF transcription contributes to the increase in TNF mRNA (Fig. 2B). Analysis of TNF mRNA stability, by actinomycin D chase methodology, showed TNF mRNA induced by IL1B to be apparently stable at 30 min post-stimulation (Fig. 2C). However, by 90 min post-IL1B, and times thereafter, TNF mRNA was reduced to ~50% of initial levels within 30 – 40 min of actinomycin D addition (Fig. 2C). In terms of these actinomycin D chase experiments, it is notable that spliced TNF mRNA levels appeared to continue rising following the addition of actinomycin D, an effect that was most apparent after 30 min of IL1B. We interpret this to mean that while actinomycin D prevents RNA polymerase II-dependent transcription, the rapid build-up of unspliced/unprocessed TNF RNA (See Fig. 2B) allows the ongoing production of mature TNF mRNA to continue briefly following the addition of actinomycin D. This interpretation is consistent with the fact that splicing and processing to produce mature mRNAs can take several minutes, or often longer (37). Thus the actinomycin D chase experiments should more accurately be considered as reflecting post-transcription RNA processing and maturation as well as mRNA degradation. Under basal conditions, TNF release into the supernatant was undetectable and at the peak of TNF mRNA expression, only low levels of TNF protein were detected (Fig. 2D, left panel). By 4 h post-IL1B, TNF release was maximal and remained at this level for up to 18 h. TNF protein inside or associated with the cells was analyzed following removal of supernatants, washing and then lysis in a soft-lysis buffer that was compatible with the ELISA. TNF protein in the cell lysates was first detected at 1 h (Fig. 2D, right panel). This was further increased at


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2 h before reaching a maximum at 4 h and then declining towards 6 h. Since soluble TNF is released into the supernatant after processing of the membrane-tethered, uncleaved, ~25kDa form of TNF by the metalloproteinase, TNF-converting enzyme (TACE) (38), we also examined the expression of uncleaved TNF, associated with the cells, by western blotting (Fig. 2E, left panel). Similar to the cell-associated TNF, detected by ELISA, uncleaved TNF was first detected at 1 h post-IL1B treatment (Fig. 2E, left panel). By 2 h, expression of uncleaved ~25kDa TNF was maximal and by 6 h, this had largely returned to basal levels.

The presence of dexamethasone co-incubated with IL1B resulted in a partial loss of TNF mRNA at all times (Fig. 2A). This effect was also observed for unspliced nuclear TNF RNA suggesting that transcriptional repression accounts for much of the repressive effect of dexamethasone on TNF mRNA (Fig. 2B). Similarly, while the decay of TNF mRNA, following 90 min of IL1B, revealed a t½ of 30 – 40 min, this was further reduced by dexamethasone, which significantly increased the loss of TNF mRNA observed at 30, 45 and 60 min post actinomycin D addition (Fig. 2C, right panels). At the level of TNF protein, dexamethasone was highly effective with near complete repression of cell-associated, uncleaved and secreted TNF, observed at all times (Fig. 2D & E). These data suggest translational, or possibly post-translational, repression by dexamethasone occurring in addition to transcriptional and post-transcriptional mechanisms of repression.

Analysis of DUSP1 expression in the presence of IL1B and dexamethasone in A549 cells – DUSP1 protein was strongly induced by IL1B and dexamethasone in A549 cells. Post-IL1B treatment, DUSP1 expression reached a peak at 1 h, before declining by 2 h and reaching basal levels by 6 h (Fig. 3A). Dexamethasone alone produced a small increase in DUSP1 at 1 h, and by 2 h this was significantly increased. In the case of IL1B plus dexamethasone treatment, DUSP1 protein was strongly induced at 1 h and declined by 2 h post treatment. However at 6 h, while IL1B-induced DUSP1 protein was undetectable, dexamethasone alone produced a significant increase in DUSP1 that was maintained in the presence of IL1B. These results are consistent with previous observations (24;28).

Analysis of ZFP36 expression in the presence of IL1B and dexamethasone in A549 cells - Western blot analysis showed ZFP36 expression to be essentially undetectable in untreated cells, but

expression, comprising of a protein doublet, was dramatically induced within 1 h of IL1B treatment (Fig.3B). Expression remained high at 2 h, but was mostly shifted to the upper, presumably phosphorylated (19;39), form of ZFP36. By 6 h, ZFP36 expression was reduced. With dexamethasone alone there was no apparent expression of ZFP36 protein at either 1 or 2 h (Fig. 3B). In the presence of IL1B, dexamethasone significantly reduced ZFP36 expression at 1 and 2 h (Fig. 3B). By 6 h, dexamethasone alone produced a small increase in ZFP36 expression and the IL1B-induced expression of ZFP36, while modest, was no longer repressed.

Effect of MAPK inhibitors on DUSP1 and ZFP36 expression - As dexamethasone, reduces MAPK activation, the effect of selective inhibitors of each of the three major MAPK pathways was tested on both IL1B-induced DUSP1 and ZFP36 expression. A549 cells were treated with maximally effective concentrations of SB203580 (10 µM), U0126 (10 µM) or JNK inhibitor 8 (JNK-IN-8) (10 µM), which inhibit the p38, (extracellular regulated kinase) ERK and JNK MAPK pathways respectively. SB203580 and JNK-IN-8 were without obvious effect on DUSP1 expression induced by IL1B at 1 h (Fig. 3C). However, UO126 consistently resulted in a partial inhibition of DUSP1 and in the presence of all three inhibitors, IL1B-induced DUSP1 protein expression was totally prevented. These data confirm the requirement for MAPK activity in the induction of DUSP1 expression by IL1B.

The presence of SB203580 completely prevented ZFP36 expression at all times, whereas the effects of U0126 or JNK-IN-8 were more modest, without significant inhibition of ZFP36 expression being achieved at any time (Fig. 3D). With all three inhibitors together there was a complete loss of IL1B-induced ZFP36 expression at all times. These data are therefore consistent with the concept that reduced MAPK activity promotes loss of ZFP36 and could lead to enhanced mRNA stability and expression of ARE-containing mRNAs (Fig. 1).

Analysis of DUSP1 expression in the presence of IL1B and dexamethasone in primary HBE cells - To validate results obtained in the A549 cells, DUSP1 mRNA and protein expression was examined in primary HBE cells. DUSP1 mRNA was strongly induced by both, IL1B and dexamethasone, at 1 h following which the mRNA expression declined rapidly by 2 h post treatment (Fig. 4A, left panel). At 18 h, while IL1B-induced DUSP1 mRNA had returned to basal levels, dexamethasone strongly induced DUSP1 mRNA. In the presence of IL1B,


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dexamethasone enhanced DUSP1 mRNA at multiple time points. However, at 18 h DUSP1 mRNA expression was primarily dexamethasone dependent, with no effect of IL1B. Likewise, DUSP1 protein was strongly induced by IL1B and IL1B plus dexamethasone (Fig. 4B, top panels). Dexamethasone alone induced DUSP1expression at 6 and 18 h post treatment and there was little or no further effect of IL1B.

Analysis of ZFP36 expression in the presence of IL1B and dexamethasone in primary HBE cells – In HBE cells, ZFP36 mRNA was strongly induced by IL1B (Fig. 4A, right panel). This was maximal at 1 h post stimulation and then declined rapidly to basal levels. Dexamethasone alone produced no changes in ZFP36 mRNA expression until 18 h and also did not affect ZFP36 expression induced by IL1B. In the case of dexamethasone co-treatment with IL1B, ZFP36 mRNA was strongly induced at 1 h and declined steeply over following 18 h post treatment. While the effect of IL1B was predominant at early time points, the late phase expression of ZFP36 mRNA at 18 h was largely dexamethasone-dependent. Similar to A549 cells, ZFP36 protein was also rapidly induced by IL1B at 1 and 2 h and declined thereafter (Fig. 4B, bottom panel). At 18 h, ZFP36 expression was variable. Dexamethasone alone did not produce any apparent increase in ZFP36 protein expression and there was no obvious effect on IL1B-induced ZFP36.

Characterization of TNF expression in the presence of IL1B and dexamethasone in HBE cells – Similar to A549 cells, treatment with IL1B rapidly increased TNF mRNA (Fig. 4C, left panel), which reached a peak at 2 h post stimulation and then declined steeply over the following 4 h. In both the A549 and HBE cells, IL1B-induced TNF were detected by real-time PCR with CT values of 22-23 cycles suggesting very similar levels of expression (data not shown). Under basal conditions, TNF release into the supernatant was undetectable and low levels of TNF protein were detected at 18 h post IL1B treatment (Fig. 4D). These data is consistent with previous findings in HBE cells showing low release of TNF in virus treated HBE cells (40). Like the A549 cells, we also examined the expression of uncleaved ~25 kDa TNF by western blotting (Fig. 4E). IL1B treatment rapidly increased uncleaved TNF expression, which was clearly detectable at 1 h, reached a peak of expression at 2 h and was lowly detectable at 6 h. Interestingly, these blots were performed in parallel with those for the A549 cells presented in Fig. 2E. Since, all the blotting and detection conditions, including exposure time, were

identical, we can conclude that uncleaved ~25 kDa TNF protein expression is similar in the A549 and HBE cells. Dexamethasone in the presence of IL1B produced a partial loss of TNF mRNA at all times (Fig. 4C right panel). At the level of TNF protein, dexamethasone also produced partial repression of uncleaved and soluble TNF at 2 and 18 h respectively (Fig. 4D & E, right panel).

A role for ZFP36 in regulating TNF mRNA expression - To assess the role of ZFP36 in the regulation of TNF mRNA expression, a siRNA-based strategy was employed. Two independent siRNA molecules directed to ZFP36, but not an unrelated LMNA siRNA, produced robust knockdown of IL1B-induced ZFP36 expression at all times tested (Fig. 5A). In respect of TNF mRNA induced by IL1B, ZFP36 knockdown had no effect at 1 h, but produced marked and significant increases in TNF mRNA expression at 2 h. While this effect was lost by 6 h post-IL1B, the data are consistent with the appearance of phosphorylated, and active, ZFP36 at 2 h. This confirms ZFP36 as a negative regulator of TNF mRNA expression. Parallel analysis of unspliced nuclear TNF RNA induced by IL1B revealed no effect of ZFP36 knockdown and this is consistent with a post-transcriptional role for ZFP36 (Fig. 5A, lower panels). To explore possible post-transcriptional regulation of TNF mRNA by ZFP36, A549 cells were treated with LMNA or ZFP36-targeting siRNAs prior to treatment with IL1B for 90 min prior to actinomycin D chase (Fig. 5B). As in Fig 2B, TNF mRNA decayed with a t1/2 of 30 – 40 min and this was not altered by the LMNA siRNA. However, both the ZFP36-targeting siRNAs significantly reduced the loss of TNF mRNA (Fig. 5B). This indicates a role for ZFP36 in the post-transcriptional regulation of IL1B-induced TNF mRNA in A549 cells.

The effect of ZFP36 knockdown was also tested on TNF mRNA and unspliced nuclear TNF RNA expression in the presence of dexamethasone and IL1B (Fig. 5A). At each time, dexamethasone produced a significant repression of TNF mRNA expression, but the level of TNF mRNA was unaltered by ZPF36 knockdown. In terms of the percentage inhibition by dexamethasone, the elevated levels of expression in the presence of the ZFP36-targeting siRNA resulted in an increased percentage repression by dexamethasone (Fig. 5A, right panels). There were no significant effects of ZFP36 knockdown observed at the level of unspliced nuclear TNF RNA (Fig. 5A, lower panels).


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Effect of DUSP1 over-expression on ZFP36 and TNF mRNA expression - Since dexamethasone induces DUSP1 expression to reduce IL1B-dependent activation of MAPKs in A549 cells (24), the effect of DUSP1 over-expression was assessed on ZFP36 protein expression. DUSP1 over-expression was confirmed following infection with DUSP1 over-expressing adenovirus, but not control (Ad-GFP) virus (Fig. 6). Substantial losses of p38, JNK and ERK MAPK phosphorylation, and therefore activity, are previously shown and were not reassessed here (24). Untreated cells showed no ZFP36 expression and this was unaltered by either adenovirus (Fig. 6). The ability of IL1B to induce ZPF36 protein was unaffected by Ad-GFP, but Ad-DUSP1 produced a near complete loss of ZFP36 expression at all times (Fig. 6). Parallel analysis of TNF mRNA revealed no effect of either virus on basal TNF mRNA expression (Fig. 6, lower panels). Likewise, Ad-GFP had no significant effect on TNF expression, but the DUSP1 over-expressing adenovirus significantly reduced TNF mRNA expression at 1 h (Fig. 6, lower panels). This repression was reduced at 2 h and, by 6 h, TNF mRNA expression appeared unaltered by Ad-DUSP1. While, these data support a role for MAPKs in the early induction of TNF mRNA expression by IL1B, it appears that DUSP1 over-expression may overcome this effect on TNF mRNA expression at longer time points.

Effect of small molecule MAPK inhibitors on TNF expression - To further explore roles for MAPK pathways in the regulation of TNF mRNA expression by IL1B, A549 cells were treated with maximally effective concentrations of SB203580 (10 µM), U0126 (10 µM) or JNK inhibitor 8 (JNK-IN-8) (10 µM). TNF mRNA expression was profoundly induced by IL1B at all times, yet this was essentially unaffected by any of the MAPK inhibitors at either 1 or 2 h post-IL1B (Fig. 7A). However, by 6 h, SB203580 significantly elevated expression of TNF mRNA relative to IL1B. Since, both dexamethasone and DUSP1 over-expression inhibit all three MAPK pathways, the combined effect of the three MAPK pathway inhibitors was assessed on TNF mRNA expression. One hour following IL1B treatment, this resulted in a substantial and significant 81.2 ± 2.4% loss of TNF mRNA (Fig. 7A, right panel). However, by 2 h post-IL1B treatment, TNF expression was 54.7 ± 6.2% of IL1B control and at 6 h there was an enhancement to 183.2 ± 22.2 %. Thus the combined inhibition of MAPKs reduced TNF mRNA expression at early time points, but that at later times this effect was both reversed and overcome.

To assess the contribution of transcriptional and post transcriptional events, the accumulation of unspliced nuclear TNF RNA and TNF mRNA half-life was assessed. In the presence of each MAPK pathway inhibitor alone, there was little or no effect on the accumulation of unspliced nuclear TNF RNA at 1 or 2 h (Fig. 7B). While there was little effect of JNK-IN-8 at 4 or 6 h post-IL1B, SB203580 and U0126 produced quite substantial, but not significant, enhancements relative to IL1B at 4 or 6 h. In the context of the three inhibitors combined, there was significant inhibition of unspliced nuclear TNF RNA induced by IL1B at 1 h, little or no effect at 2 h, followed by enhanced unspliced nuclear TNF RNA accumulation at 4 and 6 h (Fig 7B). These data suggest that MAPK-dependent transcription of TNF occurs at early times, but that MAPK inhibition leads to enhanced TNF transcription at later times.

Analysis of TNF mRNA stability (Fig. 2C) showed that following short, 30 min, IL1B treatment times, TNF mRNA appeared stable. In Fig. 7C, this is confirmed with essentially no overall loss of TNF mRNA over 60 min following actinomycin D addition. However, in the presence of SB203580 there was a reduction in the post actinomycin D accumulation combined with an overall ~50% reduction in TNF mRNA over this period. While this result may represent a mixed effect on post-transcription nuclear processing and/or mRNA stability (see above), the net effect, following transcriptional arrest, is that p38 inhibition considerably enhances loss of TNF mRNA. Pilot actinomycin D chase experiments suggest that SB203580 resulted in no effect on the loss of TNF mRNA at either 2 or 4 h post IL1B treatment (data not shown). Therefore, the effect of SB203580 was assessed on the loss of TNF mRNA after actinomycin D addition following IL1B and IL1B plus SB203580 treatments of 90, 120, 180 and 240 min. As observed at 30 min of IL1B, the presence of SB203580 significantly reduced TNF mRNA loss following 90 min IL1B stimulation, but revealed no effect following 120, 180 or 240 min treatments (Fig. 7C, right panel). These data suggest that p38 inhibition can destabilize and/or reduce nuclear RNA processing, at 30 and 90 min of IL1B, but that there was no apparent stabilization of TNF mRNA at longer treatment times. Thus MAPK inhibition results in enhanced transcription of TNF at longer time points, but may not, despite a dramatic loss of ZFP36 expression, produce an obvious stabilization of TNF mRNA. One explanation for this could be that in the absence of prior p38-dependent mRNA stabilization (occurring at 30 – 90 mins post IL1B),


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there is no possibility of any subsequent p38-dependent mRNA destabilisation. These data therefore support the existence of MAPK-dependent feed-forward mechanisms of inhibition of TNF mRNA, but do not specifically support mRNA stabilisation as a mechanism. However, since dexamethasone co-treatment with IL1B has little initial repressive effect on MAPKs, yet produces profound MAPK inhibition at longer times, it is likely that marked differences in MAPK-dependent feed-forward and feedback regulatory loops may occur. Likewise, DUSP1 knockdown in A549 cells only transiently enhanced IL1B-induced MAPK activity and this could also result in temporally distinct effects on ZFP36 and ZFP36-target genes (24).

Effect of DUSP1 knockdown on ZFP36 protein and TNF mRNA expression - To directly address the effects of reducing DUSP1 expression on TNF mRNA expression, cells were transfected with LMNA control or DUSP1-targeting siRNAs. Following IL1B treatment, DUSP1 protein was robustly induced at 1 h and this was largely blocked by the two DUSP1-targeting siRNA (Fig. 8A). Analysis of TNF mRNA revealed no effects of DUSP1 knockdown at either 1 or 2 h post-IL1B. However, by 6 h, the DUSP1-targeting siRNAs substantially and significantly reduced TNF mRNA expression relative to LMNA control siRNA (Fig. 8B). Analysis of unspliced nuclear TNF RNA also showed no significant effects of DUSP1 knockdown at 1 or 2 h, although a trend towards reduced unspliced nuclear TNF RNA was observed at 6 h. ZFP36 protein expression revealed little or no effect of DUSP1 knockdown at 1 h post-IL1B treatment (Fig. 8A). However, by 2 h post-IL1B treatment, the two DUSP1-targeting siRNAs both markedly increased expression of the upper ZFP36 band observed on western blots (Fig. 8A). These data suggest a ZFP36-dependent post-transcriptional effects could contribute to the loss of TNF mRNA following DUSP1 knockdown and is addressed in the following section.

The effect of DUSP1 knockdown induced in the presence of IL1B plus dexamethasone (Fig. 8A) was also examined on ZFP36 expression and the repression of TNF mRNA by dexamethasone. Thus, dexamethasone reduced the level of IL1B-induced ZFP36 and in the presence of the DUSP1-targeting siRNA, at 2 h, ZFP36 expression was enhanced (Fig. 8A). However, while significant repression of IL1B-induced TNF mRNA by dexamethasone was observed at all times, there was no effect of the DUSP1-targeting siRNAs. Nevertheless, at 6 h, the

large loss of IL1B-induced TNF mRNA in the presence of each DUSP1-targeting siRNA meant that there was a greatly reduced percentage repression in the presence of dexamethasone (Fig. 8B, right panels). No significant effects were apparent in respect of unspliced nuclear TNF RNA suggesting that TNF transcription rate was not affected.

Role of ZFP36 in the loss of TNF mRNA following DUSP1 knockdown - To explore a possible role for ZPF36 in mediating the enhanced loss of TNF mRNA following DUSP1 knockdown, cells were transfected with DUSP1 and ZFP36-targeting siRNAs either alone or in combination. In each case, robust knockdown of DUSP1, ZFP36, or both together was achieved (Fig. 9A). As in Fig. 5A, loss of IL1B-induced ZFP36 had little effect on TNF mRNA expression at 6 h (Fig. 9A, lower panel). Equally, targeting of DUSP1 alone resulted in a profound and significant reduction in TNF mRNA at 6 h (Fig. 9A, lower panel). This is consistent with the data in Fig. 8B. However, this repressive effect of the DUSP1 siRNAs was significantly attenuated in the additional presence of each of the ZFP36-targeting siRNAs (Fig. 9A, lower panel). This confirms a role for ZFP36 in the enhanced loss of TNF mRNA following knockdown of DUSP1.

Given that ZFP36 is an mRNA destabilizing protein, the effect of DUSP1 knockdown was assessed on TNF mRNA stability. A549 cells were therefore treated with IL1B for 2 h prior to the addition of actinomycin D (t = 0) and cells were harvested after 45 min (Fig. 9B). As shown in Figs. 2 and 5, TNF mRNA was reduced to around 50% during this 45 min time-period. However, in the presence of the DUSP1-targeting siRNAs there was significantly enhanced loss of TNF mRNA indicating that in addition to enhancing ZFP36 expression at 2 h (Fig. 8A), the DUSP1 siRNAs also reduce TNF mRNA stability. To examine the role of ZFP36 in this loss, the effect of ZFP36-targeting siRNAs were tested alone and in the presence of the DUSP1 siRNA. As previously shown (Fig. 5B), the ZFP36 siRNAs significantly reduced the loss of TNF mRNA and this confirms a role for ZFP36 in the negative regulation of TNF mRNA stability (Fig. 9B). However, in presence of both DUSP1 plus ZFP36 siRNAs, the repressive effect of the DUSP1 siRNAs were blocked and TNF mRNA decayed to the same extent as for ZFP36 knockdown alone (Fig. 9B). These data therefore support a role for ZFP36 in the enhanced decay of TNF mRNA following DUSP1 knockdown.


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The effect of combined knocking down of both DUSP1 and ZFP36 was also assessed in the presence of IL1B and IL1B plus dexamethasone at 1, 2 and 6 h. In each case, clear knockdown of DUSP1 and ZFP36 was observed (Fig. 9C). In respect of the TNF mRNA induced by IL1B, the combined knock-down produced a modest, but significant repression at 1 h, but no effect at 2 h, with, as shown in Fig. 9A, significant repression observed at 6 h (Fig. 9D, left panel). In the presence of dexamethasone, TNF mRNA was significantly repressed at all times in a manner that is consistent with Fig. 2A. At 1 and 2 h, there was no effect of the combined DUSP1 plus ZFP36 siRNAs on TNF mRNA in the presence of IL1B plus dexamethasone (Fig. 9D, left panel). However as a percentage of IL1B+LMNA at 6 h, there was significantly reduced expression of TNF mRNA induced with IL1B plus dexamethasone (16.7 ± 1.0 %) compared to 9.7 ± 0.6 & 8.3 ± 1.2 % in the presence of the DUSP1/ZFP36 combined knock-down (Fig. 9D, left panel). However, accounting for the changes in IL-1B-induced TNF mRNA with each siRNA, the loss of IL1B-induced TNF expression at 1 h produced a reduction in the percentage repression by dexamethasone, whereas there were no apparent changes in the percentage repression by dexamethasone at either 2 or 6 h (Fig. 9D, right panels).

Roles for MAPKs, DUSP1 and ZFP36 in the regulation of TNF protein expression - The above analyses address the regulation of TNF mRNA expression by DUSP1 and ZFP36 in the context of IL1B and dexamethasone. In the current section, effects on the regulation of TNF protein release are assessed. TNF protein accumulation into the supernatants was totally prevented by SB203580 and significantly inhibited by 74.0 ± 8.8 and 69.0 ± 20.2% by U0126 and JNK-IN-8, respectively (Fig. 10A). Likewise, the combination of SB203580, U0126, and JNK-IN-8 resulted in a near complete loss of TNF release. Similarly, adenoviral expression of DUSP1 also significantly inhibited TNF release, whereas the control, Ad-GFP virus, was without effect (Fig 10B). These data support a major role for MAPKs in the IL1B-induced release of TNF protein.

To examine possible roles for ZFP36 and DUSP1, TNF release was measured from the experiments shown in Figs. 5 and 8 respectively. Knockdown of ZFP36 significantly enhanced the release of TNF following IL1B treatment (Fig. 10C) and is consistent with the enhanced expression of TNF mRNA observed at 2 h in Fig 5. Conversely, targeting of DUSP1 was without obvious effect on TNF release (Fig. 10D). The effects of targeting

ZFP36 and DUSP1 were also re-confirmed in experiments where the effect of combined knock-down of ZFP36 and DUPSP1 was also examined (Fig. 10E). Thus, as shown in Fig. 10C, ZFP36 siRNA enhanced IL1B-induced TNF release and DUSP1 siRNA was without any marked effect (Fig. 10E). In the presence of ZFP36 plus DUSP1 siRNA, TNF release was more than following DUSP1 knock-down alone, but in each case this was not significantly different from the effect of targeting ZFP36 alone (Fig. 10E, left panel).

In the presence of IL1B plus dexamethasone, siRNA-targeting of ZFP36 and DUSP1 either individually (Fig. 10C & D) or in combination (Fig. 10E, middle panel) showed no significant effect on TNF release. Moreover, the percentage repression by dexamethasone was also unaltered by either treatment (Fig. 10C, D & E, right panels).

DISCUSSION Appropriate regulation of inflammatory gene

expression is central to inflammation and its resolution. Equally, understanding these processes will aid the identification of therapeutic agents that target inflammation. However, while the regulation of signal transduction and gene expression involves a myriad of signalling molecules leading to transcriptional, post-transcriptional, translational and often post-translational control of gene expression, it is routine to depict these assemblies as simple linear pathways. Using TNF biosynthesis as an example, regulation involves the integrated effect of feedback and feed-forward control loops (2;41). The current study documents interplay between the phosphatase, DUSP1, and the mRNA destabilizing protein, ZFP36, in the regulation of TNF mRNA as a model ARE-containing transcript. This network behaves in a non-linear fashion, such that modulation of individual factors may produce opposing outputs (Fig. 11A). Since, the expression of DUSP1, ZFP36 and TNF are similarly regulated by IL1B and dexamethasone in both A549 cells and primary HBE cells, our findings are likely to be physiologically and therapeutically relevant.

In a prior study, siRNA depletion of DUSP1 expression resulted, as expected for a feedback regulator of MAPKs, in enhanced activity of MAPK pathways following IL1B treatment (24). However, this effect was transient and did not necessarily produce robust increases in the mRNA expression of inflammatory genes whose expression was MAPK-dependent. More surprisingly, loss of DUSP1 reduced, relative to control, the mRNA expression for a number of inflammatory genes including;


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CCL2, CXCL3 and PTGS2 at longer (6 h) times (24). Given that; 1) such mRNAs contain AREs, 2) ZFP36 can destabilize ARE-containing mRNAs, and 3) ZFP36 is induced in A549 cells in a p38 MAPK-dependent manner, ZFP36 represents a candidate to explain this enhanced loss of inflammatory mRNAs following DUSP1 knockdown (16;41-43). We confirm, in A549 cells, that ZFP36 is induced by IL1B to reduce TNF mRNA stability and exerts feed-forward inhibition of TNF mRNA and protein expression. Furthermore, inhibition of MAPK pathways, in particular using SB203580 to inhibit p38 MAPK, prevents ZFP36 expression and increased TNF mRNA expression 6 h following IL1B treatment. Despite this, we could not document any increases in TNF mRNA stability following p38 MAPK inhibition. Rather combined MAPK inhibition reduced TNF mRNA expression at short times (1 or 2 h) and the increased TNF mRNA expression at 6 h appeared to be primarily due to enhanced transcription. These data therefore suggest the existence of, as yet uncharacterised, MAPK-dependent mechanisms that negatively regulate TNF transcription.

Despite the above, inhibition of MAPKs, in particular p38 MAPK, using kinase inhibitors or by DUSP1 over-expression, prevents MAPK activity at all times. However this may produce different functional outcomes compared to a transient modulation of MAPK activity (41). Thus, following 30 min of IL1B stimulation, TNF mRNA did not decline below the 100% level within 45 min of actinomycin D chase. This contrasts with longer IL1B treatment times, where ZFP36 is expressed and TNF mRNA decayed to 50% within 30 - 40 min of actinomycin D treatment. In the presence of the p38 MAPK inhibitor, SB203580, this apparent mRNA stabilisation did not occur and at longer times there was no destabilisation of what was presumably an already unstable transcript. This is consistent with findings in macrophage where a transient stabilization of TNF mRNA occurred 30 – 60 min post-LPS treatment, but was lost by 2 h (44). Likewise many reports indicate that p38 MAPK plays a role in the ARE-dependent stabilization of mRNAs including IL8, PTGS2 and TNF (12;42;45-47). By comparison, following silencing of DUSP1, the initial MAPK activation by IL1B occurs normally, but due to reduced feedback control, MAPK activity is not adequately down-regulated (i.e. at 1 h post IL1B-treatment in A549 cells) (24) (Fig. 11A). In this situation, we observe increased expression of ZFP36 at 2 h, followed by a subsequently enhanced loss of ARE-containing

mRNA expression at 6 h, including as is now shown, for TNF (Fig. 11A). This scheme is consistent with a role for p38 MAPK in inducing ZFP36 expression and activity to destabilize ARE-containing mRNAs such as TNF (18;48). Thus the dramatically enhanced loss of TNF mRNA following DUSP1 knockdown was not primarily associated with reduced TNF transcription, although some loss of transcription was evident and is consistent with the effect of total inhibition by the MAPK inhibitors. Rather, the enhanced loss of TNF mRNA following DUSP1 knockdown was attenuated by the further knockdown of ZFP36. This was associated with reduced TNF mRNA stability, an effect that was also attenuated by ZFP36 silencing. These data directly confirm that the loss of DUSP1 enhances ZFP36 expression to increase negative (incoherent) feed-forward control of TNF by reducing TNF mRNA stability (Fig. 11A). However, despite these data, loss of ZFP36 did not completely ablate all the effects of DUSP1 knockdown, suggesting that other non-ZFP36-dependent effects may also impact on TNF mRNA expression.

The above findings highlight a number of key points in respect of the regulation of TNF mRNA expression by glucocorticoids. Dexamethasone attenuated the accumulation of unspliced nuclear TNF RNA induced by IL1B treatment. This effect was largely reflected in TNF mRNA levels, which showed significant repression by dexamethasone at all times. While transcriptional repression is clearly important, actinomycin D chase experiments also showed dexamethasone to increase the loss of TNF mRNA. Thus, as observed for other inflammatory genes, transcriptional and post-transcriptional processes mediate the repressive effects of glucocorticoids on TNF mRNA (30;49). Furthermore, as MAPK activation is reduced by glucocorticoids in many cell types, including in A549 cells, the implication of MAPK pathways in these processes points to possible repressive mechanisms (27;29;50). However, with co-treatment, as used here, there is little repression of MAPK phosphorylation by dexamethasone at 30 min post-IL1B treatment (Fig. 11B) (24;28;35). Therefore, while inhibition of MAPKs reduces TNF transcription at this time (See Fig. 7B, right panel), this is not the mechanism for the observed repression of TNF mRNA at 30 mins by dexamethasone. Indeed one plausible explanation could be classical transrepression since the loss of IL1B-induced TNF mRNA occurs in the presence of the translational blocker, cycloheximide, and this is likely to rule out effects due to glucocorticoid-induced gene


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expression (51). Nevertheless, and irrespective of the mechanisms repressing TNF mRNA, dexamethasone induces DUSP1 expression and this inhibits MAPK activation (Fig. 11B) (24). Given that ZFP36 expression is sensitive to MAPK inhibition, dexamethasone produces a considerable reduction in ZFP36 expression (Fig. 11B). This is clearly observed at 1 and 2 h following IL1B treatment and has been previously shown in A549 cells and in LPS treated macrophages (15;16). Contrary to this, a number of groups report ZFP36 to be induced by glucocorticoids (16;31;32;52). While this glucocorticoid-inducibility seems relatively minor in A549 cells, the response may contribute towards restoring or maintaining ZFP36 expression at longer times following IL1B plus dexamethasone treatment (Fig. 11B). Nevertheless, switching off MAPKs by the dexamethasone-dependent induction of DUSP1 and other mechanisms will reduce negative feed-forward control by ZFP36 (Fig. 11B) (and potentially other factors acting on TNF transcription and mRNA stability). While this should promote TNF mRNA expression at longer times, this does not happen in the presence of glucocorticoid. Indeed, neither the silencing of DUSP1 or ZFP36, nor both together, affected the repressive effect of dexamethasone on TNF mRNA expression. Therefore a requirement for additional, non-DUSP1/non-ZFP36-dependent effector mechanisms of glucocorticoid inhibition is indicated (26;30) (Fig. 11B).

Synthesis and release of TNF protein by A549 cells into the supernatants occurs rapidly following peak mRNA expression and leads to ~100 pg/ml (~50 pg/106 cells) of TNF. Such levels are within the range (30 – 900 pg/106 cells) that are produced by mast cells, which are believed to be a physiologically relevant source of TNF (53-57). Interestingly, while in human airway epithelial cells, low nanomolar levels of TNF produce maximal responses, including cytokine release and activation of NF-κB, it is clear that low picomolar levels (~10 nM) are capable of eliciting responses, particular in the context of co-stimulants such as histamine (58). Certainly, TNF will be released in the context of multiple mediators in vivo. Furthermore, local concentrations of TNF secreted into a limited volume of fluid in the immediate vicinity of the epithelium may exceed those reached following release into a large volume of cell culture medium. Thus, it is plausible that even the low amounts of TNF produced by the primary HBEs could be sufficient to elicit paracrine effects on nearby cells. However, since membrane-tethered TNF is

biologically active, we also examined the expression of the uncleaved, ~25 kDa, TNF that is believed to exist in a membrane tethered form (59-61). Western blotting showed that expression of the 25 kDa TNF was rapidly induced by IL1B with a peak in expression at 2 h. This effect was common to both A549 and primary HBE cells and in each case peak expression was significantly and highly repressed by dexamethasone. Moreover, these levels of protein expression were similar in both cells and are consistent with the similar levels of mRNA expression observed by PCR. Furthermore, while expression of the 25 kDa TNF had largely disappeared by 6 h, in the A549 cells, we found little change in the level of TNF-associated with the cell. Therefore, while an analysis of the functional relevance are clearly beyond the scope of the current study, this membrane tethered and/or cell-associated TNF may be expected to play key roles in the rapid activation of resident or infiltrating cells.

In addition, while causing partial repression of TNF mRNA, dexamethasone produced a near complete loss of IL1B-induced TNF protein. This implies translational and/or post-translational mechanisms that produce profound inhibition of TNF expression by glucocorticoids, as has been previously suggested (62). Indeed, similar effects are reported for a number of inflammatory genes (28;63). However, even though TNF expression was strongly inhibited by MAPK inhibitors, and despite the considerable loss in mRNA expression occurring 6 h post-IL1B, silencing of DUSP1 had no effect on TNF release. This contrasts with ZFP36 silencing, which significantly increased TNF release and indicates a key role in the negative regulation of TNF translation. How this repression is achieved remains unclear, but may involve ZFP36-dependent polyA tail loss, an effect that should decrease translation efficiency and mRNA stability (10;13), both effects that are implicated in the control of TNF biosynthesis (64). Furthermore, this may account for the lack effect of the DUSP1 siRNAs. While transiently increased activation of MAPKs resulting from DUSP1 silencing should promote MAPK-dependent TNF synthesis, the corresponding increases in ZFP36 expression could counterbalance this effect (Fig. 11A). Indeed in the further presence of ZPF36 siRNA there was increased TNF protein expression suggesting that the scheme in Fig. 1 holds for TNF protein synthesis.

Turning to the repressive effect of dexamethasone, neither the ZFP36- nor the DUSP1-targeting siRNAs alone produced any change in the level of TNF released by IL1B plus dexamethasone.


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Thus neither molecule appears to be an overriding determinant for glucocorticoid repression of TNF protein. Indeed, in the presence of ZFP36-targeting siRNAs, the increased level of IL1B-induced TNF release produced by the ZFP36-targeting siRNAs meant that the percentage repression by dexamethasone actually increased (Fig. 10C). Similarly, combined knock-down of both ZFP36 and DUSP1 did not significantly alter the level of TNF release induced by IL1B plus dexamethasone or the percentage repression by dexamethasone (see Fig. 10E right panel). This confirms that additional glucocorticoid-induced repressive mechanisms must account for the repression of TNF release (Fig. 11B).

Confirmation of these findings, with respect to the effects of IL1B and dexamethasone on DUSP1, ZFP36 and TNF expression in primary HBE cells provides considerable confidence that the regulatory mechanisms described for the A549 cells are physiologically relevant to primary human airways epithelial cells. In addition, recent data in other cell lines, primary human airway smooth muscle cells and mouse models also collectively support this scheme (Fig 1) (65;66). In particular, the interaction between feedback control by DUSP1 and feed-

forward control by ZFP36 reveals why modulation of a single signalling component can lead to opposing effects on gene expression. Given an apparent loss of feed-forward control in respect of multiple inflammatory genes (24), these data illustrate the need to accurately model network behaviour in order to predict biological outcomes. Finally, ZFP36 did not completely account for the effects of DUSP1 silencing, and MAPK inhibition resulted in enhanced transcriptional responses. Thus there are likely to be a number of additional MAPK-dependent processes providing negative regulatory control of inflammatory gene expression. Such data add to the complexity of the system and confirm the need for detailed modelling to assess therapeutic strategies (41). In respect of the anti-inflammatory effects of glucocorticoids, these data suggest that neither DUSP1 nor ZFP36 have dominant repressive effects, at least on TNF expression. Importantly, the data clearly show that additional mechanisms of repression by glucocorticoids must also exist in order to maintain repression, particularly at longer times, where negative feed-forward control may be down-regulated by the early effects of glucocorticoid treatment.

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FOOTNOTES * This research was supported by: operating grants from the Canadian Institutes of Health Research (CIHR) (MOP 68828 & 125918) (RN); Lung Association of Alberta and the North West Territories and University of Calgary Studentships (SS). RN is an Alberta Innovates - Health Solutions Senior Scholar. Real-time PCR was performed by virtue of an equipment and infrastructure grant from the Canadian Fund of Innovation (CFI) and the Alberta Science and Research Authority. Disclosures: Work in the laboratory of RN was also supported by grants from AstraZeneca and GlaxoSmithKline. 1To whom correspondence should be addressed: Dr. Robert Newton, Department of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta, T2N 4N1, Canada. Tel: 001 403 210 3938. Fax: 001 403 270 8928. Email: [email protected] 2The abbreviations used are: Adenovirus serotype 5 (Ad5); Bovine serum albumin (BSA); Dual-specificity phosphatase 1 (DUSP1); Dulbecco's modified Eagle's medium (DMEM); Enzyme-linked immunosorbent assay (ELISA); Extracellular regulated kinase (ERK); Green fluorescent protein (GFP); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Interleukin (IL); Jun N-terminal kinase (JNK); Mitogen activated protein kinase (MAPK); Multiplicity of infection (MOI); Phosphate-buffered saline (PBS); Polymerase chain reaction (PCR); Tristetraprolin (ZPF36); Tumor necrosis factor α (TNF).

FIGURE LEGENDS FIGURE 1. Enhanced inflammatory gene expression by IL1B: Feed-back control by DUSP1 and feed-forward control by ZFP36. IL1B treatment results in the activation of MAPKs. This, along with the activation of other signalling pathway and inflammatory transcription factors, e.g. NF-κB and AP-1 (not shown), enhances expression of inflammatory genes (e.g. TNF), as well as the negative feed-back regulator, DUSP1, and the feed-forward regulator, ZFP36. By binding and promoting mRNA decay, and/or translational arrest, of ARE-containing mRNAs, ZPF36 is a negative, incoherent, regulator of inflammatory gene expression. Following MAPK activation, the increased expression of DUSP1 is one mechanism by which MAPK activity is restored to basal. Expression of ZFP36 depends on MAPK activation and this limits the expression of ARE-containing inflammatory genes, such as TNF. FIGURE 2. Characterization of TNF expression in the presence of IL1B and dexamethasone. A549 cells were either not treated (NT) or stimulated with IL1B (1 ng/ml) or a combination of IL1B and dexamethasone (Dex, 1 μM) as indicated. Cells were harvested at the indicated times for real-time PCR analysis of (A) TNF and GAPDH mRNA or (B) unspliced nuclear (un) TNF RNA and U6 RNA. Data (N = 4), normalized to GAPDH or U6, are plotted as means ± S.E. The effect of IL1B + dexamethasone for TNF mRNA (A right panel) (N = 12) and unspliced nuclear (un) TNF RNA (B right panel) (N = 4) is plotted as a percentage of IL1B for indicated times. Significance, relative to time matched IL1B-treated samples was tested using paired t test. *, p < 0.05. C, A549 cells were treated with IL1B (1 ng/ml) for 30, 90, 120 and 180 min. Actinomycin D (Act D, 10 μg/ml) was then added (t = 0) and the cells were harvested as indicated. RNA was extracted for real-time PCR analysis of TNF and GAPDH. Data (N = 3) were normalized to GAPDH and are plotted as a percentage of t = 0 for each treatment as means ± S.E. (left panels). A549 cells were also treated with IL1B (1 ng/ml) or IL1B plus dexamethasone (Dex, 1 μM) for 90 min. After 90 min, actinomycin D (Act D 10 μg/ml) was added (t = 0) and the cells were harvested at the indicated times for real-time PCR analysis of TNF and GAPDH. Data (n = 4) were normalized to GAPDH and are plotted as a percentage of t = 0 for each treatment as means ± S.E. (middle panel). The effect of IL1B plus dexamethasone at 45 min post Act D treatment is plotted as a percentage of t = 0 for 45 min. Significance, relative to IL1B-treated samples was tested using paired t test. *, p < 0.05 (right panel). D, Supernatants (1 ml), from the cells in A, were harvested for TNF release measurement (left panel). Alternatively, cells were harvested after 1, 2, 4 or 6 h in soft lysis buffer and total protein was prepared for the detection of cell-associated TNF via ELISA (right panel). Data (N = 4), expressed in pg/ml (for supernatant) and pg/well (for cell associated TNF), are plotted as means ± S.E. Significance, relative to time matched IL1B-treatment, was tested using paired t test. *, p < 0.05; **, p< 0.01; ***, p< 0.001. E, A549 cells were either not treated or stimulated with IL1B (1 ng/ml) for 1, 2 or 6 h (left panel) or A549 cells were either not treated or stimulated with IL1B (1 ng/ml) or a combination of IL1B and


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dexamethasone (Dex, 1 μM) for 2 h (right panel) and total protein was prepared for western blot analysis of uncleaved (~25 kDa) TNF and GAPDH. Blots representative of 4 such experiments are shown. Following densitometric analysis, data (N = 4) were normalized to GAPDH and plotted as means ± S.E. Significance, relative to non treated (left panel) or IL1B-treated (right panel) samples was tested using ANOVA with a Dunnett's post test (left panel) or paired t test (right panel) is indicated.*, p < 0.05;**, p< 0.01; ***, p< 0.001. FIGURE 3. Analysis of DUSP1 and ZFP36 expression in the presence of IL1B, MAPK inhibitors and dexamethasone. A & B, A549 cells were either not treated or stimulated with IL1B (1 ng/ml), dexamethasone (Dex, 1 μM) or a combination of the two. C & D, A549 cells were either not treated, treated with IL1B (1 ng/ml) or pre-treated with either UO126, SB203580, JNK inhibitor 8 (JNK-IN-8) or a combination of UO126, SB203580 plus JNK-IN-8 each at 10 µM for 30 min prior to IL1B stimulation. Cells were harvested at the times indicated prior to western blot analysis of DUSP1 or ZFP36 and GAPDH. All blots are representative of at least 4 such experiments are shown. Following densitometric analysis, data (N = 4) were normalized to GAPDH and plotted as means ± S.E. For A, Significance, using ANOVA with a Bonferroni's multiple comparison test is indicated. *, p < 0.05; **, p< 0.01; ***, p< 0.001. For B & D, Significance, using ANOVA with a Dunnett's post test is indicated. *, p < 0.05; **, p< 0.01. NS = non-specific band. FIGURE 4. Analysis of DUSP1, ZFP36 and TNF expression primary human bronchial epithelial cells. A, A549 cells were either not treated (NT) or stimulated with IL1B (1 ng/ml), dexamethasone (Dex, 1 μM) or a combination of the two as indicated. Cells were harvested at the indicated times for real-time PCR analysis of DUSP1, ZFP36 and GAPDH. Data (N = 4), normalized to GAPDH, are plotted as means ± S.E. Significance, using ANOVA with a Bonferroni's multiple comparison test is indicated. *, p < 0.05; **, p< 0.01; ***, p< 0.001. B, A549 cells were treated as in A and harvested at the indicated times for western blot analysis of DUSP1, ZFP36 and GAPDH. Blots representative of at least 4 such experiments are shown. NS = non-specific band. C, A549 cells were treated as in A and harvested for real-time PCR analysis of TNF and GAPDH. Data (N = 4), normalized to GAPDH, are plotted as means ± S.E. (left panel). The effect of IL1B + dexamethasone for TNF mRNA (N = 4) is plotted as a percentage of IL1B for indicated times (right panel). Significance, relative to time matched IL1B-treated samples was tested using paired t test. *, p < 0.05. D, Supernatants, from the cells in A were harvested for TNF release measurement. E, HBE cells were either not treated or stimulated with IL1B (1 ng/ml) for 1, 2 or 6 h (left panel). Alternatively cells were not treated or stimulated with IL1B (1 ng/ml) or a combination of IL1B and dexamethasone (Dex, 1 μM) for 2 h (right panel). Total protein was prepared for western blot analysis of uncleaved (~25kDa) TNF and GAPDH. Blots representative of at least 4 such experiments are shown. Following densitometric analysis, data (N = 4) were normalized to GAPDH and plotted as means ± S.E. Significance, relative to non treated (left panel) or IL1B-treated (right panel) samples was tested using paired t test.*, p < 0.05;**, p< 0.01; ***, p< 0.001. FIGURE 5. A role for ZFP36 in regulating TNF mRNA expression. A, A549 cells were incubated with LMNA (control) or ZFP36-specific siRNAs for 24 h before treatment with IL1B (1 ng/ml) or IL1B plus dexamethasone (Dex, 1 μM) as indicated. Cells were harvested at 1, 2 or 6 h and total protein was prepared for western blot analysis of ZFP36 and GAPDH. Blots representative of at least 4 such experiments are shown. NS = non-specific band. Cells were also harvested for real-time PCR analysis of TNF and GAPDH (middle panel) or unspliced nuclear (un) TNF RNA and U6 (lower panel). Data (N = 4) normalized to GAPDH or U6, were expressed as a percentage of LMNA siRNA plus IL1B-stimulated for each time and are plotted as means ± S.E. Significance was tested using ANOVA with a Newman-Keul multiple comparison test. Significance for specific comparisons are indicated. *, p < 0.05; **, p< 0.01; ***, p< 0.001. The effect of IL1B plus Dex expressed as a percentage of IL1B for each of the three individual siRNAs is plotted as a mean ± S.E. (right panels). B, A549 cells were incubated with LMNA or ZFP36-specific siRNAs for 24 h before being treated with IL1B (1 ng/ml) for 90 min. Actinomycin D (Act D, 10 μg/ml) was then added (t = 0) and cells were harvested at the indicated times. RNA was extracted for real-time PCR analysis of TNF and GAPDH. Data (N = 4), normalized to GAPDH, are plotted as a percentage of t = 0 for each treatment as means ± S.E. Significance, relative to time matched IL1B-treated samples was tested using paired t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001, (left panel). The effect of IL1B plus LMNA siRNA and IL1B plus ZFP36 siRNAs at 45 min post Act D treatment is plotted as a percentage of t = 0 for 45 min. Significance, relative to IL1B-treated samples was tested using ANOVA with a Dunnett's post test. **, p < 0.01, (right panel).


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FIGURE 6. Effect of DUSP1 over-expression on ZFP36 and TNF mRNA expression. A549 cells were either not infected or infected with Ad5-DUSP1 or Ad5-GFP (control) at a MOI of 10 for 24 h before IL1B treatment (1 ng/ml). Cells were harvested after 1, 2 or 6 h and total protein prepared for western blot analysis of DUSP1, ZFP36 and GAPDH. Blots representative of at least 4 such experiments are shown. NS = non-specific band. Following densitometric analysis for ZFP36, data (N = 4) were normalized to GAPDH and plotted as means ± S.E. (middle panels). Significance, using ANOVA with a Dunnett's post test is indicated. *, p < 0.05. Cells were also harvested for real-time PCR analysis of TNF and GAPDH (lower panels). Data (N = 4), normalized to GAPDH, are plotted as means ± S.E. Significance relative to time-matched IL1B and Ad5-GFP-treated samples, was tested using ANOVA with a Bonferroni post test. **, p< 0.01; ***, p< 0.001. FIGURE 7. Effect of MAPK inhibitors on TNF expression. A, A549 cells were either not treated (NT), treated with IL1B (1 ng/ml) or pre-treated with either UO126, SB203580, JNK inhibitor 8 (JNK-IN-8) or a combination of UO126, SB203580 plus JNK-IN-8 each at 10 µM for 30 min prior to IL1B stimulation. Cells were harvested after 1, 2 or 6 h for real-time PCR analysis of TNF and GAPDH. Data (N = 4) were normalized to GAPDH and are plotted as means ± S.E. The effect of IL1B + MAPK inhibitors for TNF mRNA is plotted as a percentage of IL1B at the indicated times. B, RNA from the experiments in A were subjected to real-time PCR analysis of unspliced nuclear (un) TNF RNA and U6. Data (N = 4) were normalized to U6 and are plotted as means ± S.E. The effect of IL1B + MAPK inhibitors for unspliced nuclear (un) TNF mRNA is plotted as a percentage of IL1B at the indicated times. For both A & B, significance, relative to either non-treated or IL1B-treated samples was tested using ANOVA with a Bonferroni post test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, A549 cells were treated with IL1B (1 ng/ml) or pre-treated for 30 min with SB203580 at 10 µM prior to IL1B stimulation for 30 min. Following this (t = 0), actinomycin D (Act D, 10 μg/ml) was added and cells were harvested at the indicated times. RNA was extracted for real-time PCR analysis of TNF and GAPDH. Data (N = 4) normalized to GAPDH, are plotted as a percentage of t = 0 for each treatment as means ± S.E. (left panel). Data following 45 min of Act D treatment are shown (right panel). A549 cells were also treated with IL1B or IL1B + SB203580 at 10 µM for 90, 120, 180 and 240 min, prior to the addition of Act D (t = 0) for 45 min and analyzed as above (Right panels). Significance, relative to time matched IL1B-treated samples was tested using paired t test. *, p < 0.05. FIGURE 8. Effect of DUSP1 knockdown on ZFP36 and TNF expression. A, A549 cells were incubated with LMNA (control) or DUSP1-specific siRNAs for 24 h before treatment with IL1B (1 ng/ml) or IL1B plus dexamethasone (Dex, 1 μM) as indicated. Cells were harvested at 1, 2 or 6 h and total protein was prepared for western blot analysis of DUSP1, ZFP36 and GAPDH. Blots representative of at least 7 – 9 such experiments are shown. NS = non-specific band. B, Cells were treated as in A and harvested after 1, 2 or 6 h for real-time PCR analysis of TNF and GAPDH (upper left panel) or unspliced nuclear (un) TNF and U6 (lower left panel). Data (N = 4 - 8), normalized to GAPDH (for TNF mRNA) or U6 (for unspliced nuclear (un) TNF RNA), were expressed as a percentage of LMNA siRNA plus IL1B-stimulated for each time and are plotted as means ± S.E. Significance was tested using ANOVA with a Newman-Keul multiple comparison test. Significance for specific comparisons is indicated. **, p< 0.01; ***, p< 0.001. The percent IL1B plus dexamethasone/IL1B for the LMNA siRNA was also plotted and compared with that for each of the DUSP1-specific siRNAs using ANOVA with a Dunnett's post test. **, p< 0.01 (right panel). FIGURE 9. Role of ZFP36 in the enhanced loss of TNF mRNA following DUSP1 knockdown. A, A549 cells were incubated with LMNA (control) or DUSP1- and/or ZFP36-specific siRNAs for 24 h before being treated with IL1B (1 ng/ml). While not indicated, LMNA siRNA was added to all the treatment groups except for the combination siRNA treatments to maintain a constant concentration of siRNAs across all the treatments. Cells were harvested after 1 h and total proteins were prepared for western blot analysis of DUSP1, ZFP36 and GAPDH. Blots representative of at least 4 such experiments are shown (top panel). NS = non specific band. Cells were also harvested after 6 h for real-time PCR analysis of TNF and GAPDH. Data (N = 4) normalized to GAPDH were expressed as a percentage of LMNA siRNA plus IL1B and plotted as means ± S.E. Significance was tested using ANOVA with a Newman-Keul multiple comparison test (lower panel). B, A549 cells were treated as in A before being treated with IL1B (1 ng/ml) for 2 h. Following this (t = 0), actinomycin D (Act D, 10 μg/ml) was added and cells were harvested after 45 min. RNA was extracted for real-time PCR analysis of TNF and GAPDH. Data (n = 6) normalized to GAPDH, are plotted as a percentage of t = 0 for each treatment as means


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± S.E. For both A & B, significance between: LMNA control siRNA plus IL1B and each of the DUSP1 and/or ZFP36 targeting siRNAs plus IL1B is shown by #. Other comparisons are specifically indicated. */#, p < 0.05; ##, p< 0.01; ***/###, p< 0.001. C, A549 cells were incubated with LMNA (control) or DUSP1 and ZFP36-specific siRNAs for 24 h before being treated with IL1B (1 ng/ml) or IL1B plus dexamethasone (Dex, 1 μM) as indicated. Cells were harvested after 1 h and total proteins were prepared for western blot analysis of DUSP1, ZFP36 and GAPDH. Blots representative of at least 4 such experiments are shown. NS = non specific band. D, A549 cells were treated as in C and harvested after 1, 2 or 6 h for real-time PCR analysis of TNF and GAPDH. Data (N = 4) normalized to GAPDH were expressed as a percentage of LMNA siRNA plus IL1B-stimulated for each time and are plotted as means ± S.E. Significance was tested using ANOVA with a Newman-Keul multiple comparison test. Significance for specific comparisons are indicated.*, p < 0.05; **, p< 0.01; ***, p< 0.001 (left panel).The percent IL1B plus dexamethasone/IL1B for the LMNA siRNA was also plotted and compared with that for each of the DUSP1 and ZFP36-specific siRNAs using ANOVA with a Dunnett's post test.*, p < 0.05 (right panel). FIGURE 10. Roles for MAPKs, DUSP1 and ZFP36 in the regulation of TNF protein expression. A, A549 cells were either not treated, treated with IL1B (1 ng/ml) or pre-treated with either UO126, SB203580, JNK inhibitor 8 (JNK-IN-8) or a combination of UO126, SB203580 plus JNK-IN-8 each at 10 µM for 30 min prior to IL1B stimulation. B, A549 cells were either not infected or infected with Ad5-DUSP1 or Ad5-GFP (control) at a MOI of 10 for 24 h before IL1B treatment (1 ng/ml). For both A & B, cells supernatants were harvested after 6 h for TNF release measurement. Data (N = 4) expressed in pg/ml are plotted as means ± S.E. Significance, using ANOVA with a Bonferroni's multiple comparison test is indicated. *, p < 0.05; **, p< 0.01; ***, p< 0.001. C, D & E, A549 cells were incubated with LMNA (control) or DUSP1-and/or ZFP36-specific siRNAs for 24 h before being treated with IL1B (1 ng/ml) or IL1B plus dexamethasone (Dex, 1 μM) as indicated. Cells supernatants were harvested after 6 h for TNF release measurement. Data (N = 4) expressed in pg/ml are plotted as means ± S.E. Significance, using ANOVA with a Newman-Keul multiple comparison test is indicated. *, p < 0.05; **, p< 0.01; ***, p< 0.001 (left panels). In each case, the percent IL1B plus dexamethasone/IL1B for the LMNA siRNA was also plotted and compared with that for each of the DUSP1- and ZFP36-specific siRNAs using ANOVA with a Dunnett's post test (right panels). For E (left panel): LMNA siRNA was added in all the treatment groups except for the combination siRNA treatments in order to maintain a constant siRNA concentration across all the treatments. FIGURE 11. Regulation of TNF gene expression following DUSP1 silencing and glucocorticoid treatment. Schematics representing possible regulatory networks are shown along with summarized data from the current and a prior manuscript (24). A, With reduced DUSP1 expression there is reduced negative feedback control of MAPKs leading to enhanced MAPK activity (at 1 h) (bold) at the times where DUSP1 expression would have been elevated. This could promote enhanced TNF expression. However, enhanced MAPK activity enhances expression of ZFP36 (at 2 h) (bold), which may decrease TNF expression (at 6 h). Actual expression of TNF is dependent on the temporal interplay of these competing regulatory processes. B, In the presence of glucocorticoid co-treatment, DUSP1 expression is enhanced (bold) and promotes inactivation of MAPKs (grey). Loss of MAPK activity reduces the expression of feed-forward negative control genes, such as ZFP36. However, ZFP36 is also modestly induced by glucocorticoid alone (in brackets) and this may help maintain feed-forward control. Given the net loss of IL1B-induced ZFP36 expression in the presence of glucocorticoids (at 1 and 2 h), additional glucocorticoid effectors must exist to maintain repression of TNF (at later times). Repression of MAPKs is also maintained by non-DUSP1-dependent mechanisms. Positive signalling/expression (blue) is represented by arrows. Negative effects are indicated (red) by lines ending in a T-bar. Time course expression of phospho-p38, DUSP1 and ZFP36 protein and TNF mRNA in the presence of IL1B and IL1B plus DUSP1 siRNA (A) or IL1B plus dexamethasone (B) is shown. The protein expression data for DUSP1 and ZFP36 were generated following densitometric analysis of western blots showed in figure 3. For phospho-p38, western blots were taken from Shah et al. 2014 (24). For phospho-p38, DUSP1, ZFP36 and TNF, at each time, the effect of IL1B plus Dex expressed as a percentage of IL1B is plotted as a mean (B, right panels).


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F

(pg/w

ell)

Cell associated

NT IL1B IL1B+Dex

Supernatant

1 2 4 60

50

100

* *

**

time (h)

IL1B

+D

ex

(% o

f IL

1B

at

each tim

e)

0

50

100

150 90 min

0

100

200

300 30 min

0 15 30 450

50

100

150

time (min)

120 min

0 15 30 450

50

100

150 180 min

time (min)

0.0

0.5

1.0

1.5**

***

0

50

100 *


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Fig. 3

0.0

1.5

3.0 * *

ZFP36

GAPDH

1 2

+ + + + - - + + - - + +

6

+ +

- - + +

time (h)

Dex

IL1B

ZF

P36

/GA

PD

H

pro

tein

NS

B

0

1

2

3

******

***

***

**

***

****

***

******

**

DUSP1

GAPDH

2 6

+ + + + - - + + - - + +

time (h) ½ 1

Dex + + + + IL1B - - + + - - + +

A

NS

DU

SP

1

/GA

PD

H

pro

tein

1

+ +

+ + + + - + + + + +

time (h)

JNK-IN-8 UO126 SB203580 IL1B

2

+ +

+ +

+ + - + + + + +

ZFP36

GAPDH

ZF

P36

/GA

PD

H

pro

tein

NS

6

+ +

+ +

+ + - + + + + +

0.0

0.4

0.8*

**

****

D

1

+ +

+ + + + - + + + + +

time (h)


NS DUSP1

GAPDH

C


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Fig. 4

A

DUSP1

GAPDH

2 6

+ + + + - - + + - - + +

time (h) ½ 1

Dex + + + + IL1B - - + + - - + +

18

+ +

- - + +

NS ZFP36

GAPDH

B

NT

Dex

IL1B

IL1B+Dex

0

1

2

3

****

*** ***********

***

*****

**

time (h)

DU

SP

1

/GA

PD

H

mR

NA

0.0

2.5

5.0

**

******* *

**

*

time (h)

ZF

P3

6

/GA

PD

H

mR

NA

½ 1 2 6 18 ½ 1 2 6 18

C time (h) 2

Dex + IL1B - + +

D

0

2

4

time (h)

TN

F

rele

ase

(pg/m

l)

time (h) 18

Dex + + IL1B - - + +

E

0

2

4

**

***

Uncleaved TNF

GAPDH

TN

F

/GA

PD

H

pro

tein

time (h) 1 1 2 6

IL1B - + + +

TN

F

/GA

PD

H

pro

tein

(% IL1B

)

Uncleaved TNF

GAPDH

0

50

100**

0 5 10 150.0

0.3

0.6

0.9

time (h)

TN

F/G

AP

DH

mR

NA

NT Dex

IL1B IL1B+Dex

IL1B

+D

ex

(% o

f IL

1B

at

each tim

e)

1 2 6 180

100

200

* *

time (h)


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Fig. 5

0

100

200

300

******

******

******

*** ***

***

* ***

A

ZFP36

GAPDH

time (h) 1

ZFP36 siRNA 2 + + ZFP36 siRNA 1 + + LMNA siRNA + + Dex + + + IL1B - + + + + + +

2

+ +

+ + + +

+ + + - + + + + + +

6

+ +

+ + + +

+ + + - + + + + + +

TN

F/G

AP

DH

mR

NA

(%IL

1B

+

LM

NA

siR

NA

)

unT

NF

/U6

RN

A

(%IL

1B

+

LM

NA

siR

NA

)

0

50

100

150

NS

time (h) 1

ZFP36 siRNA 2 + ZFP36 siRNA 1 + LMNA siRNA + IL1B+Dex + + +

2

+

+ +

+ + +

6

+

+ +

+ + +

0

50

100

Eff

ect of

Dex o

n

TN

F/G

AP

DH

(% IL1B

Rele

vant

siR

NA

)

0

50

100

Eff

ect of

Dex o

n

unT

NF

/U6

(% IL1B

Rele

vant

siR

NA

)

B

0 20 40 600

50

100

150

time (min)0 20 40 60

0

50

100

150

***

time (min)0 20 40 60

0

50

100

150

****

time (min)

LMNA siRNA ZFP36 siRNA1 ZFP36 siRNA2

TN

F/G

AP

DH

mR

NA

(% t =

0)

TN

F/G

AP

DH

mR

NA

(% t =

0)

0

25

50

75 ****


ZFP36 siRNA 2 + ZFP36 siRNA 1 + LMNA siRNA + IL1B + + +

IL1B

IL1B+siRNA


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Fig. 6

0.0

0.5

1.0

1.5

*

*

2

+ +

+ + - - - + + +

time (h) 1

Ad-DUSP1 + + Ad-GFP + + IL1B - - - + + +

ZFP36

GAPDH

6

+ +

+ + - - - + + +

ZF

P36

/GA

PD

H

pro

tein

DUSP1

GAPDH T

NF

/GA

PD

H

mR

NA

(% o

f IL

1B

at each tim

e)

0

50

100

150 ******

*****

NS


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0 2 4 60.0

0.5

1.0

1.5

****

***

NT

IL1B

time (h)

0 2 4 60.0

0.5

1.0 NT

IL1B

***

***

**time (h)

Fig. 7

A

B unT

NF

/U6

RN

A

(%

of IL

1B

at

each tim

e)

unT

NF

/U6

RN

A

TN

F/G

AP

DH

mR

NA

(%

of IL

1B

at

each tim

e)

TN

F/G

AP

DH

mR

NA

C

0 20 40 600

100

200

300

**

* *

time (min)

IL1B

IL1B +

SB203580

TN

F/G

AP

DH

mR

NA

(% t =

0)

0

25

50*

0

25

50

0

25

50

0

25

50

0

50

100

150*

IL1B treatment time (min) 30 90 120 180 240

SB203580 + + + + + IL1B + + + + + + + + + +

TN

F/G

AP

DH

mR

NA

(% t =

0)

1 2 60

100

200SB203580

*

time (h)1 2 6

0

100

200UO126

time (h)1 2 6

0

100

200JNK-IN-8

time (h)1 2 6

0

100

200SB+UO+J8

****

*

time (h)

1 2 4 60

200

400SB203580

time (h)1 2 4 6

0

200

400UO126

time (h)1 2 4 6

0

200

400JNK-IN-8

time (h)1 2 4 6

0

200

400SB+UO+J8

*

*

time (h)


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Fig. 8

A

DUSP1

GAPDH

ZFP36

GAPDH

time (h) 1

DUSP1 siRNA 2 + + DUSP1 siRNA 1 + + LMNA siRNA + + Dex + + + IL1B - + + + + + +

2

+ +

+ + + +

+ + + - + + + + + +

6

+ +

+ + + +

+ + + - + + + + + +

NS

NS

time (h) 1


2

+ +

+ + + +

+ + + - + + + + + +

6

+ +

+ + + +

+ + + - + + + + + +

0

50

100

150 ****

B

TN

F/G

AP

DH

mR

NA

(%IL

1B

+

LM

NA

siR

NA

)

unT

NF

/U6

RN

A

(%IL

1B

+

LM

NA

siR

NA

)

time (h) 1

DUSP1siRNA 2 + DUSP1siRNA 1 + LMNA siRNA + IL1B+Dex + + +

2

+

+ +

+ + +

6

+

+ +

+ + +

0

50

100

***

Eff

ect of

Dex o

n

TN

F/G

AP

DH

(% IL1B

Rele

vant

siR

NA

)

0

50

100

Eff

ect of

Dex o

n

unT

NF

/U6

(% IL1B

Rele

vant

siR

NA

)

0

100

200 ***

**** ***

****

******

***


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Fig. 9

DUSP1

GAPDH

ZFP36

GAPDH

time (h) 1

ZFP36 siRNA 2 + + DUSP1 siRNA 2 + + ZFP36 siRNA 1 + + DUSP1 siRNA 1 + + IL1B - + + + + + + +

A

NS

NS

TN

F/G

AP

DH

mR

NA

(%IL

1B

+LM

NA

siR

NA

)

0

50

100

150*

*

######

#####

6 h

C

ZFP36

GAPDH

DUSP1

GAPDH

NS

time (h) 1

DUSP1 + ZFP36 siRNA 2 + + DUSP1 + ZPF36 siRNA 1 + + LMNA siRNA + + Dex + + + IL1B - + + + + + +

NS

B

0

50

100

150***

# #

### ######

###

***


ZFP36 siRNA 2 + + ZFP36 siRNA 1 + + DUSP1 siRNA 2 + + DUSP1 siRNA 1 + + IL1B + + + + + + +

TN

F/G

AP

DH

mR

NA

(% t =

0)

D

0

50

100*

0

50

100

0

50

100

0

50

100

150

*

******

** ***

1 h

0

50

100

150***

****** 2 h

0

50

100

150***

***

******

**

***

**6 h

TN

F/G

AP

DH

mR

NA

(%IL

1B

+LM

NA

siR

NA

)

DUSP1 + ZFP36 siRNA 2 + DUSP1 + ZFP36 siRNA 1 + LMNA siRNA + IL1B+Dex + + +

Eff

ect of D

ex o

n

TN

F/G

AP

DH

(% IL1B

R

ele

vant

siR

NA

)



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Fig. 10

A

TN

F

rele

ase

(pg/m

l)

0

100

200

***

******

***

6

+ +

+ + + + - + + + + +

time (h)


time (h) 6

Ad-DUSP1 + + Ad-GFP + + IL-1B - - - + + +

TN

F

rele

ase

(pg/m

l)

0

50

100*

**

B

time (h) 6


0

4

8

12

****

**

TN

F

rele

ase

(pg/m

l)

time (h) 6

ZFP36 siRNA 2 + + ZFP36 siRNA 1 + + LMNA siRNA + + Dex + + + IL1B - + + + + + +

0

20

40

60***

******

*

***

TN

F

rele

ase

(pg/m

l)

C time (h) 6

ZFP36 siRNA 2 + ZFP36 siRNA 1 + LMNA siRNA + IL1B+Dex + + +

0

50

100

Eff

ect of

Dex o

n

rele

ase

(% IL1B

Rele

vant

siR

NA

)

time (h) 6

DUSP1 siRNA 2 + DUSP1 siRNA 1 + LMNA siRNA + IL1B+Dex + + +

0

50

100

Eff

ect of

Dex o

n

rele

ase

(% IL1B

Rele

vant

siR

NA

)

E time (h) 6

DUSP1 siRNA 2 + + DUSP1 siRNA 1 + + ZFP36 siRNA 2 + + ZFP36 siRNA 1 + + IL1B - + + + + + + +

TN

F

rele

ase

(pg/m

l)

0

20

40 ****

**

0

50

100

Eff

ect of

Dex o

n

rele

ase

(% IL1B

Rele

vant

siR

NA

)

0

40

80***

**

***

***

***

TN

F

rele

ase

(pg/m

l)

time (h) 6


time (h) 6

DUSP1 + ZFP36 siRNA 2 + DUSP1 + ZFP36 siRNA 1 + LMNA siRNA + IL1B+Dex + + +

D


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Fig. 11

IL1B

MAPK DUSP1 Feed-forward

control (ZFP36)

TNF gene

expression

GC

GC

Other GC

effectors

IL1B + glucocorticoid

Other GC

effectors

B

0 2 4 6

0

100

200

300TNF mRNA

time (h)

0

50

100

150ZFP36 protein

0

100

200 DUSP1 protein

0

350

700 Phosho-p38

Gene/G

AP

DH

% 1

h IL1B

NT IL1B IL1B+Dex

0

50

100

150ZFP36 protein

IL1B

MAPK

Feed-forward

control by ZFP36

IL1B treatment (No DUSP1)

TNF gene

expression

A

Gene/G

AP

DH

% 1

h IL1B

+Lsi

0

50

100

150

200Phospho-p38

DUSP1 siRNA

0

100

200ZFP36 protein

TN

F/G

AP

DH

% IL1B

+Lsi

NT IL1B IL1B+siRNA

0 2 4 6

0

50

100

150TNF mRNA

time (h)

IL1B

+D

ex

(% o

f IL

1B

at each tim

e)

0

50

100

150

Phospho-p38

0

250

500 DUSP1 protein

1 2 4 6

0

50

100

TNF mRNA

time (h)

¼ ½ 1 2 6

1 2 6


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NewtonSuharsh Shah, Mahmoud M. Mostafa, Andrew McWhae, Suzanne L. Traves and Robert

glucocorticoidsmitogen-activated protein kinase phosphatase, DUSP1: implications for regulation by

Negative feed-forward control of TNF by tristetraprolin (ZFP36) is limited by the

published online November 6, 2015J. Biol. Chem.

10.1074/jbc.M115.697599Access the most updated version of this article at doi:

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negative regulation of tnf by zfp36 1 negative feed-forward

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